Tethering cysteine residues using cyclic disulfides

ABSTRACT

Described herein are compounds and methods for tethering proteins. For example, dimers of proteins, including SOD1 and DJ-1, are described, where the dimers are formed by the covalent bonding of a cysteine on the first monomer to a cysteine on the second monomer via a cyclic disulfide linker. The covalently attached dimers exhibit increased stabilization.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/440,978, filed May 6, 2015, which is a National Stage application ofPCT/US13/070239, filed Nov. 15, 2013, which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/726,776,filed Nov. 15, 2012.

BACKGROUND OF THE INVENTION

Many therapeutic molecules form covalent bonds with cysteine residues ontheir protein targets. The mechanisms of the majority of these moleculeswere either elucidated long after development or are not fullyunderstood. Recent successful drug discovery efforts, however, moved tostructure-based design. These require both an accurate structural modelof the target protein and a high-specificity ligand.

One third of therapeutic molecules, including many blockbuster drugs,form covalent bonds with their targets. These electrophilic drugsgenerally bond to a nucleophilic amino acid, often serine or cysteine,on a target protein. Aspirin and penicillin (and their many derivatives)acylate serines and numerous drugs form covalent bonds with specificcysteines. These therapeutic agents are effective despite the potentialfor off-target reactions with hundreds of highly reactive, nucleophilicresidues, which are often required for the function of essentialproteins. A worst case scenario for reaction with the “wrong”nucleophile is nerve gases (e.g., Sarin, intravenous LD₅₀ ˜30 μg/kg),which covalently modify the active site serine of acetylcholineesterase. On the other hand, comparable toxicity has been harnessed toselectively target cancer cells-bortezomib/Velcade (LD₁₀₀<250 μg/kg)selectively modifies an active site threonine of the proteasome.Unintended reaction with a highly reactive nucleophile isn't necessarilydisastrous—it has led to a drug. The disulfide-containing substance,disulfiram, was intended to treat parasitic infections, but when testedon humans gave severe “hangover” symptoms upon alcohol consumption.Years after its therapeutic use began, this compound, dubbed antabuse,was found to bind the highly reactive active site cysteines of alcoholdehydrogenase. Nevertheless, the paucity of therapeutic suicideinhibitors to most human proteases, which (unlike viral proteases) havenumerous homologues with identical off-target catalytic sites, has beenattributed to off-target nucleophiles.

With effective covalent drugs, off-target binding tends to be offset byselectivity for the target and the enhanced potency inherent toirreversible inhibition. The uncanny specificities of cysteine-bindingtherapeutics involve elegant and usually serendipitous chemistry. Thegastroesophageal reflux disease drugs (GERD, e.g., omeprazole/Prilosec™lansoprazole/Prevacid™, etc.) use a cyclic sulphenamide to irreversiblybind a cysteine residue of the proton pump of the intestinal lumen.These benzolamide-derivative prodrugs require protonation of a low pKapyridine nitrogen (pKa <4.5) for activation and sequestration. They areneutral, inactive, and permeable, but are activated upon encounteringthe pH ˜0.8 parietal cell canaliculus, which contains their target(i.e., the proton pump). Here, they accumulate at 1000-fold higherconcentrations. While the chemical basis of proton-mediated accumulationof omeprazole was appreciated, if not designed, the elegant sulfur-basedchemistry behind activation and binding of a target cysteine wasserendipitous.

The antithrombosis factors clopidogrel/Plavix™, ticlopide/Ticlid, etc.are also prodrugs. Activation by cytochrome P450 enzymes results in thescission of a ring carbon-sulfur bond, creating a sulfhydryl group thatcan then form a disulfide bond with its target cysteine on the adenosinediphosphate (ADP) chemoreceptor P2Y₁₂. In addition to increasedspecificity for its target, which it permanently inactivates, the activemetabolite has improved plasma protein binding characteristics. Thethrombosis drugs had their beginnings in functional assays, andfortunately animal studies, because the active metabolite is notproduced in most cell-based assays. Both their mechanism of action andtarget were unknown at the time of discovery.

More recent compounds employing sulfhydryl moieties were rationallydesigned. Dacomitinib, afatinib, and neratinib are EGFR kinaseinhibitors with a high-affinity, nucleotide-analogue moiety thatreversibly binds the ATP-binding pockets of numerous kinases and asecond moiety designed to covalently bond with a non-conserved cysteine(present in EGFR but not its homologues). The electrophilic moiety ispurposefully a low-reactivity acrylamide to minimize off-targetreactions. A related chemical approach used low-reactivity,acrylamide-based, electrophiles to target non-conserved (in humans) andnon-catalytic-residue cysteine of the HCV NS3/4A viral protease (HCVP).

In sum, all known approaches either minimize the exposure of highlyreactive electrophiles (“hiding” reactive sulfur in disulfides or inrings), or minimize the reactivity of exposed electrophiles (usingacrylamide adducts). Unfortunately, however, the specificity ofsulphenamides depends upon an acidic environment (pH<4.5) found only inthe intestinal lumen, and the specificity of therapeutics employingreactive sulfhydryl groups is poorly understood. A few therapeuticmolecules were obtained by rationally attaching low-reactivityelectrophiles to high affinity and specificity moieties. Unfortunately,compounds with high affinity and specificity tend to appear in the finalstages of a drug development effort making this approach best suited forimproving existing specificity.

There exists a need for a strategy for conferring specificity to drugsthat target cysteine, in general, but pairs of cysteine, in particular.

SUMMARY OF THE INVENTION

Representative Methods of the Invention

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound of Formula I or a compound of Formula II with afirst protein and a second protein under conditions suitable forcross-linking the first protein to the second protein, therebycross-linking the first protein to the second protein,

wherein

the first protein comprises a first cysteine residue;

the second protein comprises a second cysteine residue;

the compound of Formula I is

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

the compound of Formula II is

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound with a first protein and a second protein underconditions suitable for cross-linking the first protein to the secondprotein, thereby cross-linking the first protein to the second protein,

wherein

the first protein and the second protein have at least 90% sequencehomology;

the first protein and the second protein are SOD-1 or DJ-1; and

the compound is a compound of Formula I

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl.

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound with a first protein and a second protein underconditions suitable for cross-linking the first protein to the secondprotein, thereby cross-linking the first protein to the second protein,

wherein

the first protein and the second protein have at least 90% sequencehomology;

the first protein and the second protein are SOD-1 or DJ-1; and

the compound is a compound of Formula II

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to a method of treating orpreventing a condition, comprising the step of

administering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula I or a compound of Formula II,

wherein

the compound of Formula I is

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

the compound of Formula II is

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to a compound of Formula I

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl.

One aspect of the invention is a stabilized superoxide dismutaseanalogue, wherein said analogue has a tertiary structure and comprises afirst SOD1 monomer and a second SOD1 monomer; wherein the first SOD1monomer comprises a first cysteine residue; the second SOD1 monomercomprises a second cysteine residue; the first cysteine residue isconnected to the second cysteine residue by a connection; and theconnection is a connection of Formula III or Formula IV:

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4;

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

One aspect of the invention is a stabilized DJ-1 analogue, wherein saidanalogue has a tertiary structure and comprises a first DJ-1 monomer anda second DJ-1 monomer; wherein the first DJ-1 monomer comprises a firstcysteine residue; the second DJ-1 monomer comprises a second cysteineresidue; the first cysteine residue is connected to the second cysteineresidue by a connection; and the connection is a connection of FormulaIII or Formula IV:

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4;

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a) a potential reaction mechanism where a cyclicdisulfide reacts with the cysteine of one monomer and the resultingthiolate can then react with, for example, a hydrogen peroxide-modifiedthiolate on the other monomer (Isaac, et al. Chemical Science 2012); b)side-chains of Cys53′s in the dimer interface of DJ-1 (PDB: 3SF8) (SEQID NO:1), demonstrating their close spacing (Premkumar, et al. J.Struct. Biol. 2011, 176, 414). The appearance of Cys111's in the dimerinterface of SOD1 is very similar.

FIG. 2 depicts a preliminary LC-ESI-IonTrap-MS screen of cyclicdisulfides that identifies multiple compounds that form covalent SOD1dimers. a) Cyclic disulfides identified in our preliminary screen thatform covalent-linked SOD1 dimers. b) Deconvoluted LC-MS spectra of SOD1with no compound (top) and with 1-oxo-1,2-dithiane (bottom).

FIG. 3 depicts a preliminary LC-ESI-IonTrap-MS screen of cyclicdisulfides that identifies compounds that form covalent DJ-1 dimers atCys53 and increase the thermal stability of DJ-1. a) NSC72268 wasidentified as a specific covalent dimerizer of DJ-1. b) Deconvolutedspectra of untreated DJ-1 (top) and NSC72268-treated DJ-1. c)MALDI-TOF-MS spectra showing a detected ion specific forNSC56224-treated DJ-1 corresponding to two trypsin digest fragments ofDJ-1 containing Cys53 (underlined) linked by NSC56224. d) NSC56224 andNSC72268 increase the measured denaturation temperature of DJ-1 relativeto untreated DJ-1 (N=3, error bars not shown but standard deviation isless than thickness of lines).

FIG. 4 depicts examples of cyclic disulfide compounds that are able toform covalent dimers of SOD1.

FIG. 5 depicts a dithiol that is able to form covalent dimers of SOD1.

FIG. 6 depicts examples of cyclic disulfide compounds that are able toform covalent dimers of DJ-1.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The covalent attachment of molecules can be used to affect proteinstructure and function. Covalently attached molecules can be used toinhibit, promote activity, stabilize, and destabilize proteins andpeptides. One problem is encoding adequate specificity in covalentbinders for the intended target. In certain embodiments, the inventionrelates to chemical tools, cyclic disulfides, that target pairs ofcysteine residues and significantly enhance the specificity for pairs ofcysteine over lone cysteine residues. In addition to augmenting currentapproaches to rational design, cyclic disulfides offer a launching pointfor compound optimization for novel targets. Whereas previous approachesto covalent modification tended to be devoted to inactivation of anenzyme, cyclic disulfides are also amenable to protein (includingenzyme) stabilization. We apply cyclic-disulfides to the stabilizationof two proteins involved in neurodegenerative disease, Cu/Zn-SOD1, whichis involved in amyotrophic lateral sclerosis and potentially Parkinson'sand Alzheimer's, and DJ-1, which is involved in Parkinson's.

SOD1 and Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis is a progressive neurodegenerative diseasecaused by death of motor neurons in the brain and spinal cord. Theoverall median survival from onset of symptoms ranges between 2-3 yearsfor cases with bulbar onset to 3-5 years for cases with limb onset.Lifetime risk of ALS is 1/400 to 1/1000 with a median annual incidenceof 1.89 and a median prevalence of 5.2 per 100,000 each year. Thereexists no cure for ALS and the only FDA-approved treatment for ALS,riluzole (Rilutek), prolongs median survival by a mere 2-3 months whentaken for an eighteen month duration. Thus, novel therapeutic strategiesfor ALS continue to be crucial. Approximately ten percent of ALS isfamilial (fALS) and approximately twenty percent of fALS cases arecaused by autosomal dominant mutations in the ubiquitously expressedprotein SOD1. Over 100 SOD1 mutations have been identified which arelinked with fALS and it is thought they confer a toxic gain of function.As the clinical phenotypes of patients with various fALS SOD1-associatedmutations are more alike than different, and all appear to cause thedeath of motor neurons, it has been hypothesized that mutations sharecommon properties and mechanisms of cytotoxicity. In addition to causingtwenty percent of fALS, SOD1 may be playing a role in sporadic ALS.Evidence is emerging that a subset of sporadic ALS is characterized byunfolded WT SOD1, and that oxidatively modified SOD1 slows axonaltransport to a similar extent to the G93A SOD1 variant. Numerous otherreports have also implicated oxidized/misfolded WT SOD1 as beingcytotoxic and/or related to sporadic ALS.

One prevailing hypothesis for the mechanism of the toxicity ofALS-associated SOD1 variants involves dimer destabilization anddissociation into monomers, which then nucleate the formation ofhigher-order aggregates. ALS-associated variants of SOD1, such as G85R,are found as monomers in ALS patients and a number of modifications,including loss of Cu or Zn, cleavage of the native, intramoleculardisulfide, oxidation, glutathionylation, and fALS-associated mutation,predispose the SOD1 dimer to dissociate. X-ray crystal structures ofboth A4V, and to a lesser extent I113T, yeast two-hybrid analysis ofH46R, A4V, and H48Q, dissociation of G85R, G93R, E100G, and I113T bychaotrophs, and molecular dynamics simulations are all consistent withthis hypothesis; mutations and modifications destabilize dimerformation. Furthermore, destabilization of dimer formation has beenfound to be reversible through both the tethering of subunits with agenetically engineered inter-subunit disulfide and the use of smallmolecules and this prevents protein aggregation. Thus, dimerstabilization is being pursued as a therapeutic strategy.

SOD1 dimers contain two cysteine residues at the dimer interface whosesulfhydryl groups are approximately nine angstroms apart. Thesesulfhydryl groups can be targeted by maleimide cross-linkers which leadto strong stabilization of ALS-associated SOD1 dimers. Surprisingly,while cross-linking at sulfhydryl groups by the maleimides occurred bypredicted maleimide-mediated mechanisms, for the maleimidedithio-bismaleimidoethane (DTME), it was found that stabilization of theSOD1 dimer possibly occurred through both maleimide interaction with thesulfhydryl group of Cys111 on one SOD1 monomer as well asthiol-disulfide exchange between the disulfide spacer of DTME and thesulfhydryl group of the Cys111 on the second SOD1 monomer.Unfortunately, maleimides are highly irritating locally and have an LD₅₀in mice of 9 mg/kg with renal, hepatic, neurologic and hematologictoxicities as the principal effects of the drug in this species.Therefore, in certain embodiments, the invention relates to thediscovery of molecules that can cross-link SOD1 dimers in order to fullyassess small molecule-mediated covalent dimer formation of SOD1 as atherapeutic strategy for ALS.

DJ-1 and Parkinson's Disease (PD)

The progressive neurodegenerative disorder PD is characterized by theloss of dopaminergic neurons in the substania nigra pars compacta andα-synuclein-rich protein deposits known as Lewy bodies. A variety ofpharmacological treatment options exist for the early-stage symptoms ofPD as the patient becomes functionally impaired. However, as the diseaseprogresses, all of these agents, which primarily treat the symptoms ofPD, become ineffective as fewer dopaminergic neurons survive. Thus, asthe ability to slow the progression of the disease remains elusive,novel directions in therapeutic development are necessary to furthercombat PD.

While the majority (>90%) of PD cases are idiopathic, mutations inPARK7, encoding the 189-amino acid homodimeric protein DJ-1, are knownto be a rare cause of autosomal recessive early-onset Parkinson disease.Some evidence also indicates polymorphisms in PARK7 confer risk insporadic PD patients. Biochemical and cell culture analysis of PD-linkedvariants of DJ-1 suggest a number of mechanisms through which structuraldefects, including loss of stability and dimer formation, may lead to aloss-of-function that is associated with PD pathogenicity, such asreduced ability to prevent α-synuclein aggregation, deficiency inoxidative stress-dependent RNA-binding activity, reduced ability to actas a neuroprotective transcriptional co-activator, and increasedsensitivity to oxidative stress-induced cell death related tomitochondrial defects. In additional to recessive PD-related mutants ofDJ-1 being implicated in disease, analysis of DJ-1 in the frontal cortexof patients with sporadic PD and Alzheimer's disease reveal that acidicisoforms of monomeric DJ-1 and basic isoforms of SDS-resistant dimericDJ-1 selectively accumulate in these diseases, with DJ-1 irreversiblyoxidized by carbonylation as well as by methionine oxidation tomethionine sulfone. Over-oxidation of DJ-1 has been found to producestructural destabilization similar to PD-related mutations, suggestingthat dysfunctional DJ-1 due to aberrant modifications could be a causeof sporadic neurodegenerative cases.

Just as loss of DJ-1 function appears to contribute to the etiology ofPD, evidence suggests that enhancement of DJ-1 function could compensatefor other causes of PD. DJ-1 protects against degeneration of nigraldopaminergic neurons in PD rat models involving both 6-hydroxydopamineand rotenone treatment. Viral-mediated DJ-1 overexpression in the MPTPmouse model has also proved efficacious in reducing nigral dopamineneuron loss. Likewise, pharmacological upregulation of DJ-1 with thehistone deacetylase inhibitor phenylbutyrate rescues cells fromoxidative stress and mutant α-synuclein toxicity, as well as protectsdopaminergic neurons from MPTP-induced neurotoxicity and preventsage-related motor and cognitive decline in mice with diffuse Lewy bodydisease. Thus, enhancement of DJ-1 activity could serve as a therapeuticstrategy in a possibly wide variety of PD cases. Previously, in silicomethods have been used to identify potential small molecule bindingsites on DJ-1 and for identifying small molecules capable of interactingwith DJ-1 and modulating its oxidation state that have neuroprotectiveeffects in vivo. Amazingly, DJ-1 dimers have a set of cysteines,Cys53's, spaced closely together around the dimer interface similar toSOD1, suggesting covalent dimerization at these cysteines might also bepossible. Enhancing dimer formation of DJ-1 at these cysteines has beenconceptually demonstrated via an engineered disulfide bond produced by aV51C mutation in DJ-1, which rescues structural and functional defectsdue to modification and mutations. Interestingly, highly reactivedopamine quinones have been observed to form covalent dimers of DJ-1 viaCys53, a putative natural mechanism of covalent dimer stabilization ofDJ-1. However, Cys53s in DJ-1 are more closely spaced than Cys111s inSOD1, hindering previous attempts to covalently dimerize DJ-1 usingmaleimide crosslinkers.

Dithiols and Cyclic Disulfides as Covalent Dimerizers and Therapeutics

With the observation that DTME was able to cross-link SOD1 monomerspartially through thiol-disulfide exchange, we proposed that cyclicdisulfides may provide an alternative for covalently dimerizing bothSOD1 and DJ-1 through their respective closely spaced dimer interfacecysteines. Theoretically, a cyclic disulfide could undergothiol-disulfide exchange with the cysteine of one SOD1/DJ-1 monomer,leaving a free thiolate to react with the remaining monomer (FIG. 1).Dithiols might also be capable of forming covalent dimers if the thiolgroups are properly spaced and appropriately reactive. In agreement withthis hypothesis, we present several cyclic disulfides and a dithioldiscovered in preliminary screens that are capable of covalentlydimerizing SOD1 and/or DJ-1. Several cyclic disulfides are already knownto be safe for human consumption and/or are potential therapeuticsincluding 4,5-dihydroxy-1,2-dithiane and α-lipoic acid (ALA), suggestingmore cyclic disulfides might also make feasible drug developmentcandidates. Furthermore, while there remains a common apprehensionagainst the development of covalently acting drugs for fear ofindiscriminate reactivity, numerous examples exist of such drugs thathave excellent safety records, with proven techniques existing tosystematically rank lead compounds based on selectivity.

Representative Methods of the Invention

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound of Formula I or a compound of Formula II with afirst protein and a second protein under conditions suitable forcross-linking the first protein to the second protein, therebycross-linking the first protein to the second protein,

wherein

the first protein comprises a first cysteine residue;

the second protein comprises a second cysteine residue;

the compound of Formula I is

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

the compound of Formula II is

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound with a first protein and a second protein underconditions suitable for cross-linking the first protein to the secondprotein, thereby cross-linking the first protein to the second protein,

wherein

the first protein and the second protein have at least 90% sequencehomology;

the first protein and the second protein are SOD-1 or DJ-1; and

the compound is a compound of Formula I

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is S.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is S═O.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein Y is S(═O)₂.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein n is 1 or 2.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein n is 1.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein n is 2.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is selected from the groupconsisting of

In certain embodiments, the invention relates to a method comprising thestep of

contacting a compound with a first protein and a second protein underconditions suitable for cross-linking the first protein to the secondprotein, thereby cross-linking the first protein to the second protein,

wherein

the first protein and the second protein have at least 90% sequencehomology;

the first protein and the second protein are SOD-1 or DJ-1; and

the compound is a compound of Formula II

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the R″ form a six-membered ring. Incertain embodiments, the invention relates to any one of theaforementioned methods, wherein the R″ form an aromatic ring. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the R″ form a six-membered aromatic ring.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first protein and the second proteinhave at least 95% sequence homology.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first protein and the second proteinhave at least 98% sequence homology.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first protein and the second proteinhave at least 99% sequence homology.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first protein and the second proteinare wild type SOD-1.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the first protein and the second proteinare wild type DJ-1.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is in vitro. In certainembodiments, the invention relates to any one of the aforementionedmethods, wherein the first protein and the second protein are in a cell.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is a method of inhibiting theactivity of the first protein or the second protein.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is a method of increasing theactivity of the first protein or a second protein.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is a method of stabilizingthe first protein or the second protein.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the method is a method of destabilizingthe first protein or the second protein.

In certain embodiments, the invention relates to a method of treating orpreventing a condition, comprising the step of

administering to a subject in need thereof a therapeutically effectiveamount of a compound of Formula I or a compound of Formula II,

wherein

the compound of Formula I is

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

the compound of Formula II is

wherein

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and Y is S.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and Y is S═O.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and Y is S(═O)₂.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and n is 1 or 2.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and n is 1.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of Formula I;and n is 2.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is the compound is acompound of Formula I; and the compound is selected from the groupconsisting of

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of FormulaII; and the R″ form a six-membered ring. In certain embodiments, theinvention relates to any one of the aforementioned methods, wherein thecompound is a compound of Formula II; and the R″ form an aromatic ring.In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of FormulaII; and the R″ form a six-membered aromatic ring.

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the compound is a compound of FormulaII; and the compound is

In certain embodiments, the invention relates to any one of theaforementioned methods, wherein the condition is ALS, Parkinson'sdisease, or Alzheimer's disease.

Representative Compounds of the Invention

In certain embodiments, the invention relates to a compound of Formula I

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4; and

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is S.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is S═O.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein Y is S(═O)₂.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1 or 2.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 1.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein n is 2.

In certain embodiments, the invention relates to any one of theaforementioned compounds, wherein the proviso that the compound is notselected from the group consisting of

Representative Analogues of the Invention

One aspect of the invention is a stabilized superoxide dismutaseanalogue, wherein said analogue has a tertiary structure and comprises afirst SOD1 monomer and a second SOD1 monomer; wherein the first SOD1monomer comprises a first cysteine residue; the second SOD1 monomercomprises a second cysteine residue; the first cysteine residue isconnected to the second cysteine residue by a connection; and theconnection is a connection of Formula III or Formula IV:

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4;

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the tertiary structure issubstantially the same as the wild-type superoxide dismutase enzyme.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the sequence homology of said firstSOD1 monomer and said second SOD1 monomer is greater than or equal toabout 85%.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said first SOD1 monomer and saidsecond SOD1 monomer have substantially the same amino acid sequence.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the first SOD1 monomer of saidanalogue is the wild-type sequence or comprises a mutation selected fromthe group consisting of G93A, G85R, D90A, A4V, E100G, H46R, C6G, andI113T.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the second SOD1 monomer of saidanalogue is the wild-type sequence or comprises a mutation selected fromthe group consisting of G93A, G85R, D90A, A4V, E100G, H46R, C6G, andI113T.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue retains at least 90%activity of the wild-type superoxide dismutase enzyme up to atemperature of about 75° C.

One aspect of the invention is a stabilized DJ-1 analogue, wherein saidanalogue has a tertiary structure and comprises a first DJ-1 monomer anda second DJ-1 monomer; wherein the first DJ-1 monomer comprises a firstcysteine residue; the second DJ-1 monomer comprises a second cysteineresidue; the first cysteine residue is connected to the second cysteineresidue by a connection; and the connection is a connection of FormulaIII or Formula IV:

wherein

Y is S, S═O, or S(═O)₂;

n is 0, 1, 2, 3, or 4;

R is independently selected from the group consisting of —H, —OH, —NH₂,—NHR′, —N(R′)₂, alkyl, —OMs, —OTs, —OTf, and —CO₂H; or any two geminal Rgroups, taken together, form an imine; or any two vicinal R groups,taken together, form a ring; wherein any alkyl or imine may besubstituted with a carbamide, a carboxylate, or a hydroxyl; and

R′ is alkyl or aryl; and

R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the tertiary structure issubstantially the same as the wild-type DJ-1.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein the sequence homology of said firstDJ-1 monomer and said second DJ-1 monomer is greater than or equal toabout 85%.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said first DJ-1 monomer and saidsecond DJ-1 monomer have substantially the same amino acid sequence.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue retains at least 90%activity of the wild-type DJ-1 up to a temperature of about 75° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization from about 10° C. to about 60° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization from about 20° C. to about 40° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization from about 15° C. to about 25° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization from about 30° C. to about 50° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization about 20° C.

In certain embodiments, the present invention relates to any one of theaforementioned analogues, wherein said analogue is increased instabilization about 40° C.

Definitions

The term “analogue” refers to a molecule substantially similar infunction to SOD1 protein or a fragment thereof.

The terms “percent (%) amino acid sequence identity” or “percent aminoacid sequence homology” or “percent (%) identical” as used herein withrespect to a reference polypeptide is defined as the percentage of aminoacid residues in a candidate polypeptide sequence that are identicalwith the amino acid residues in the reference polypeptide sequence afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, without considering anyconservative substitutions as part of the sequence identity. Alignmentfor the purpose of determining percent amino acid sequence identity canbe achieved by various techniques known in the art, for instance, usingpublicly available computer software such as ALIGN or Megalign(DNASTAR). Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the peptide sequence beingused in the comparison. For example, in the context of the presentinvention, an analogue of SOD1 is said to share “substantial homology”with SOD1 if the amino acid sequence of said analogue is at least about85%, at least about 90%, at least about 95%, or at least about 99%identical to wild-type.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals, substantiallynon-pyrogenic, without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, not injurious to thepatient, and substantially non-pyrogenic. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include: (1)sugars, such as lactose, glucose, and sucrose; (2) starches, such ascorn starch and potato starch; (3) cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;and (21) other non-toxic compatible substances employed inpharmaceutical formulations. In certain embodiments, pharmaceuticalcompositions of the present invention are non-pyrogenic, i.e., do notinduce significant temperature elevations when administered to apatient.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

A “therapeutically effective amount” of a compound, e.g., such as apolypeptide or peptide analogue of the present invention, with respectto use in treatment, refers to an amount of the polypeptide or peptidein a preparation which, when administered as part of a desired dosageregimen (to a mammal, preferably a human) alleviates a symptom,ameliorates a condition, or slows the onset of disease conditionsaccording to clinically acceptable standards for the disorder orcondition to be treated or the cosmetic purpose, e.g., at a reasonablebenefit/risk ratio applicable to any medical treatment.

The terms “prophylactic” or “therapeutic” treatment are art-recognizedand includes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 General Materials and Methods

Cross-Linking and Western Blots

WtSOD1 or wtDJ-1 was incubated with 5-25 mM DTT for approximately 20minutes and either buffer exchanged using Amicon Ultra-4 centrifugalspin concentrators (MWCo 10K) or using reversed phase chromatography(ZIPTIP, Millipore, Inc). Samples cleaned by ZIPTIPs were also subjectedto incubation with 5 mM EDTA. SOD1 samples that were buffer exchangedusing Amicon concentrators were exchanged into in HPLC water, whereasZIPTIP samples were further exchanged after ZIPTIP into PBS, pH 7.4 orHPLC water. DTT-reduced SOD1 or DJ-1 was incubated at a 1:1 (20 μM:20 μMor 10 μM:10 μM) or 1:3 (20 μM:60 μM or 10 μM:30 μM) ratio of protein tocross-linker.

A variety of cross-linkers were used. Cross-linking was achieved byincubating the reaction in either PBS pH 7.4 or water at roomtemperature for 1 hour. After an hour the reactions were analyzed on a15% SDS-PAGE gel with a non-cross-linked control, transferred tonitrocellulose membrane and western blotted using a polyclonal antibodyto SOD1 or DJ-1. Repeated in triplicate.

In addition, DTME is a cleavable sulfhydryl-sulfhydryl cross-linkingagent. Therefore, a cross-linking reaction containing 1:1 molar rationof wtSOD1 or wtDJ-1 to DTME was performed at room temperature for onehour. After cross-linking, the reaction was split in half and half ofthe sample was run in a sample buffer containing DTT (reducing) and theother half in one containing no DTT (non-reducing). These samples alongwith non-cross-linked controls were then analyzed on a 15% SDS PAGE geland western blotted as above.

Matrix Assisted Laser Desorption Ionization (MALDI)-Time of Flight (TOF)

wtSOD1 or wtDJ-1 was cross-linked as below. After cross-linking, 1 μL ofsample was spotted on a MALDI target containing 1 μl of matrix, 20 mg/mLsinipic acid, and analyzed on a Bruker Daltonics Microflex. The MALDIwas calibrated each time using a high molecular weight proteincalibration standard, Protein Calibration Standard I (Bruker Daltonics).The MALDI-TOF was operated in linear mode using a laser power of between72-90%. MALDI-TOF spectra were of cross-linked and non-cross-linkedsamples were analyzed using FlexAnalysis software (Bruker Daltonics).Repeated in triplicate.

Example 2

LC-MS screen of cyclic disulfides with SOD1 reveals small molecules thatcovalently link SOD1 dimers. Compounds being evaluated were dissolvedand incubated with recombinant human WT SOD1 (SEQ ID NO:2). Reactionswere analyzed by LC-ESI-IonTrap-MS on a HCT Ultra ion trap (BrukerDaltonics, Billerica, MA, USA). The resulting data was examined usingDataAnalysis 3.4 (Bruker Daltonics Inc., Billerica, MA, USA). Massspectra were averaged across the retention times corresponding to whenSOD1 was found to be eluting and Maximum Entropy Deconvolution wasapplied to the resulting average mass spectrum in order to determine themolecular weight of the uncharged species detected. Significantdithiol-and cyclic disulfide-mediated covalent dimer formation has beenobserved with multiple different compounds. For example, the changes inmass of the covalently linked SOD1 dimers observed suggest both1,2-dithiolane-4,4-dimethanol and 1-oxo-1,2-dithiane (FIG. 2a ) arecapable of covalently dimerizing SOD1. Of note, 1-oxo-1,2-dithiane wasable to cross-link the majority of SOD1 monomers in the sample (FIG. 2b) and the mass of the cross-linked SOD1 dimer corresponds to the loss ofone water molecule after the addition of 1-oxo-1,2-dithiane to a SOD1dimer. 1-oxo-1,2-dithiane (NSC56224) and its analogues have beenpartially characterized previously for their ability to attackretroviral zinc fingers. Identified compounds can be found in FIG. 4 andFIG. 5.

Example 3

LC-MS screen of cyclic disulfides with DJ-1 reveals molecules thatcovalently link DJ-1 dimers. Using the same method as described forSOD1, compounds were screened against WT DJ-1. In addition to NSC56224again being identified as a covalent dimerizer of DJ-1, NSC72268 wasidentified as a specific DJ-1 covalent dimerizer (FIG. 3a,b ). DigestingNSC56224-linked DJ-1 with trypsin following by MALDI-TOF-MS confirmedNSC56224 covalently linked DJ-1 dimers at Cys53 (FIG. 3c ). BothNSC56224 and NSC72268 were found to increase the denaturationtemperature of DJ-1 measured with differential scanning fluorimetry(FIG. 3d ) (Niesen et al., 2007), suggesting covalent dimerizationincreased DJ-1 thermal stability. NSC72268 and NSC56224 are shown inFIG. 6.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A stabilized superoxide dismutase dimer or a stabilizedDJ-1 dimer, wherein said stabilized superoxide dismutase dimer has atertiary structure and comprises a first superoxide dismutase (SOD1)monomer and a second SOD1 monomer; wherein the first SOD1 monomercomprises a first cysteine residue; the second SOD1 monomer comprises asecond cysteine residue, and wherein said first SOD1 monomer and saidsecond SOD1 monomer is at least 85% to SEQ ID NO:2; or said stabilizedDJ-1 dimer has a tertiary structure and comprises a first DJ-1 monomerand a second DJ-1 monomer; wherein the first DJ-1 monomer comprises afirst cysteine residue; the second DJ-1 monomer comprises a secondcysteine residue, and wherein said first DJ-1 monomer and said secondDJ-1 monomer is at least 85% to SEQ ID NO:1; and wherein the firstcysteine residue is connected to the second cysteine residue by aconnection; and the connection is a connection of Formula Ill or FormulaIV:

wherein Y is S, S═O, or S(═O)₂; n is 0, 1, 2, 3, or 4; R isindependently selected from the group consisting of —H,—OH, —NH₂, —NHR′,—N(R′)₂, alkyl,—OMs, —OTs, —OTf, and —CO₂H; or any two geminal R groups,taken together, form an imine; or any two vicinal R groups, takentogether, form a ring; wherein any alkyl or imine may be substitutedwith a carbamide, a carboxylate, or a hydroxyl; and R′ is alkyl or aryl;and R″ is —H, alkyl, or aryl, or both R″, taken together, form a ring;wherein any alkyl, aryl, or ring may be substituted with —OH, alkyl, orhalo.
 2. The stabilized dimer of claim 1, wherein said dimer is thestabilized superoxide dismutase dimer.
 3. The stabilized dimer of claim1, wherein said dimer is the stabilized DJ-1 dimer.
 4. The stabilizedsuperoxide dismutase dimer of claim 2, wherein said first SOD1 monomerand said second SOD1 monomer are different in amino acid sequence. 5.The stabilized superoxide dismutase dimer of claim 2, wherein said firstSOD1 monomer and said second SOD1 monomer have the same amino acidsequence.
 6. The stabilized superoxide dismutase dimer of claim 2,wherein the first SOD1 monomer of said dimer is the wild-type sequence(SEQ ID NO:2).
 7. The stabilized superoxide dismutase dimer of claim 2,wherein the first SOD1 monomer of said dimer comprises a mutationselected from the group consisting of G93A, G85R, D90A, A4V, E100G,H46R, C6G, and I113T of SEQ ID NO:2.
 8. The stabilized superoxidedismutase dimer of claim 7, wherein the second SOD1 monomer of saiddimer is the wild-type sequence (SEQ ID NO:2).
 9. The stabilizedsuperoxide dismutase dimer of claim 7, wherein the second SOD1 monomerof said dimer comprises a mutation selected from the group consisting ofG93A, G85R, D90A, A4V, E100G, H46R, C6G, and I113T of SEQ ID NO:2. 10.The stabilized superoxide dismutase dimer of claim 2, wherein said dimerretains at least 90% activity of the wild-type superoxide dismutaseenzyme (SEQ ID NO:2) up to a temperature of about 75° C.
 11. Thestabilized DJ-1 dimer of claim 3, wherein said first DJ-1 monomer andsaid second DJ-1 monomer are different in amino acid sequence.
 12. Thestabilized DJ-1dimer of claim 3, wherein said first DJ-1 monomer andsaid second DJ-1 monomer have the same amino acid sequence.
 13. Thestabilized DJ-1 dimer of claim 1, wherein said dimer retains at least90% activity of the wild-type DJ-1 up to a temperature of about 75° C.14. The stabilized superoxide dismutase dimer of claim 2, wherein theconnection is a connection of Formula III:

wherein Y is S, n is 2, and R is H.
 15. The stabilized DJ-1 dimer-ofclaim 3, wherein the first DJ-1 monomer of said dimer is the wild typeDJ-1 (SEQ ID NO:1).
 16. The stabilized superoxide dismutase dimer ofclaim 3, wherein the connection is a connection of Formula III:

wherein Y is S, n is 2, and R is H.